Long-lasting activity-dependent alterations in the strength of synaptic connections are thought to be essential substrates of memory. Long-term potentiation (LTP) of synaptic transmission has been heavily studied, long-term depression (LTD) of synapse strength much less so. Prolonged low-frequency synaptic stimulation elicits robust LTD of synaptic strength. Our previous studies suggest that LTD consists of multiple, distinct cascades, one dependent on the actions of the intercellular messenger nitric oxide (NO), production of cyclic GMP and inhibition of cyclic AMP-dependent protein kinase, which acts on presynaptic terminals to persistently reduce transmitter release. Induction of presynaptic LTD can depend on activation of N-methyl-D-aspartate receptors (NMDAR), or on co-activation of group I and II metabotropic glutamate receptors. Using two-photon laser scanning microscopy of FM1-43 release from hippocampal synaptic terminals, we have shown that NMDAR-dependent presynaptic LTD is associated with a selective reduction in release from the rapidly-recycling vesicle pool (RRP). In this renewal application, we propose studies that employ electrophysiological recording and two-photon confocal fluorescence imaging techniques in in vitro hippocampal slices to answer the following questions: (1) Are NMDAR-dependent and mGLuR-dependent forms of LTD induced by different biochemical pathways? (2) What are the effects of NMDAR- and mGluR-dependent forms of LTD on kiss-and-run" release, vesicle recycling and pool exchange rates visualized with FM1-43 and in excitatory and inhibitory synapses in synaptopHluorin-expressing mice?, and 3) What is the role of G[unreadable]? binding the synaptic vesicle proteins or voltage-dependent calcium channels in presynaptic terminals in NMDAR- versus mGluR-dependent LTD? There is a probable role for LTD in memory processing, and impairments in LTD may contribute to pathologies of memory storage such as Alzheimer's Disease. LTD-like dampening of neuronal excitation could be important in preventing epileptic seizures and reducing excitotoxic neuronal injury. The long-term regulation of transmitter release has far-reaching importance to normal synaptic processing dynamics, and controlling pathologic glutamate release during injury or disease. PUBLIC HEALTH RELEVANCE: Mechanisms by which brain electrical activity persistently reduces connection strengths between neurons are poorly understood, and are likely to be critical to how the brain stores information and regulates overall electrical excitability. We propose experiments to use fluorescent indicators that sense presynaptic neurotransmitter release to uncover the mechanisms of this reduction in synaptic strength, called "long-term depression". This work is potentially vital to our understanding of normal memory storage, and diseases ranging from Alzheimer's to epilepsy, in which synaptic plasticity may be dysfunctional.